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PMID:11124266

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Citation

Cahill, CM, Tzivion, G, Nasrin, N, Ogg, S, Dore, J, Ruvkun, G and Alexander-Bridges, M (2001) Phosphatidylinositol 3-kinase signaling inhibits DAF-16 DNA binding and function via 14-3-3-dependent and 14-3-3-independent pathways. J. Biol. Chem. 276:13402-10

Abstract

In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C. elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction of DAF-16 with its cognate DNA site. Elimination of the DAF-16/1433 association by mutation of the AKT/14-3-3 sites in DAF-16, prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3 association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity. Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, our results demonstrate at least two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA binding and transcription function.

Links

PubMed Online version:10.1074/jbc.M010042200

Keywords

14-3-3 Proteins; Amino Acid Sequence; Animals; Binding Sites; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Consensus Sequence; DNA/metabolism; DNA-Binding Proteins/metabolism; Humans; MAP Kinase Signaling System/physiology; Mammals; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Phosphatidylinositol 3-Kinases/metabolism; Phosphorylation; Protein-Serine-Threonine Kinases; Protein-Tyrosine Kinases/metabolism; Proto-Oncogene Proteins/metabolism; Proto-Oncogene Proteins c-akt; Recombinant Fusion Proteins/metabolism; Recombinant Proteins/metabolism; Transcription Factors/metabolism; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase/metabolism

Significance

Annotations

Gene product Qualifier GO ID GO term name Evidence Code with/from Aspect Notes Status


See also

References

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